Detergent composition containing proteolytic enzyme elaborated by thermoactinomyces vulgaris atcc 15734

ABSTRACT

A DETERGENT COMPOSITION USEFUL FOR ORDINARY HOUSEHOLD LAUNDRY AND DISHWASHING APPLICATIONS COMPOSED OF A SYNTHETIC DETERGENT AND AN EFFECTIVE AMOUNT OF A PROTEOLYTIC ENZYME ELABORATED BY THERMOACTINOMYCES VULGARIS ATCC 15734.

United States Patent US. Cl. 252-135 4 Claims ABSTRACT OF THE DISCLOSURE A detergent composition useful for ordinary household laundry and dishwashing applications composed of a synthetic detergent and an effective amount of a proteolytic enzyme elaborated by Thermoactinomyces vulgaris ATCC 15734.

FIELD OF THE INVENTION This invention relates to detergent compositions which contain an enzyme ingredient in addition to a synthetic detergent ingredient. Specific embodiments of this invention are directed to detergent compositions which contain, in addition to a detergent and an enzyme, such additional ingredients are builders, water, bleaching agents and the like.

BACKGROUND OF THE INVENTION Detergent compositions containing enzymes have been known for many years. See, for example, US. Pat. 1,882,- 270, Frelinghausen, granted Oct. 11, 1932, German Auslegeschrift 14,296, Leidholdt, dated Jan. 6, 1958, and US. Pat. 3,451,935, Roald and deOude, granted June 24, 1969.

Enzymes aid in the detergency process by attacking soils and stains found on soiled substrates such as ordinary household laundry clothing and fabrics as well as hard surface applications such as washing soiled dishes, glassware, and metal instruments and appliances. Soils and stains, such as food stains, grass stains, etc., are decomposed, degraded or otherwise altered by enzymes and thereby rendered more easily removable. Enzyme-containing laundry compositions can be used either in a soaking or pre-wash product designed to prepare soiled fabrics for more effective cleaning when the fabrics are subjected to conventional laundering, or an enzyme-containing composition can be used as a component of a complete detergent formulation containing conventional cleaning ingredients such as synthetic detergents, builders, bleaching agents and the like. For use in dishwashing and other hard surface or light-duty household cleaning applications, enzyme-containing compositions comprise primarily a synthetic detergent, an enzyme ingredient and, optionally, lesser amounts of builders than are present in heavy-duty compositions.

Not all enzymes are capable of being satisfactorily incorporated into detergent compositions. Many enzymes are readily denatured by detergents and the other ingredients generally employed in preparing useful detergent compositions. Other enzymes are too unstable in aqueous systems and, therefore, cannot be used in washing and cleaning processes. In addition, to be useful enzymes must be resistant to degradation by hot or cold Water temperatures, and have sustained enzyme activity in different pHs such as the highly alkaline systems of built detergent compositions.

The need exists for different types of enzymes which can provide detergent compositions having overall satisfactory performance or which can provide detergent compositions having unique performance characteristics under a combination of the several conditions mentioned above.

3,707,504 Patented Dec. 26, 1972 ice SUMMARY OF THE INVENTION It has now been discovered that a proteolytic enzyme elaborated by T hermoactinomyces vulgaris ATCC 15734 is useful in detergent compositions of the type described above.

One embodiment, therefore, of this invention is an unbuilt detergent composition consisting essentially of a water-soluble synthetic detergent and an effective amount of a proteolytic enzyme elaborated by Thermoactinomyces vulgaris ATCC 15734. By effective amount is intended an amount of enzyme which provides a satisfactory level of enzyme activity units as described hereinafter to accomplish a desired type of cleaning. This invention can be practiced to provide useful compositions having different levels of activity. Compositions intended for soaking and Washing heavily soiled fabrics, for instance, generally require a larger amount of enzymatic activity than a composition intended for washing lightly soiled fabrics.

Unbuilt compositions as just described can be formulated into granular, tablet, or liquid products. In the latter event, water is the typical liquid carrier but such materials as hydrotropes and organic materials such as alcohols can be used provided the activity of the enzyme is not disturbed.

According to another embodiment of this invention, a highly useful built detergent composition is provided consisting essentially of a synthetic detergent, a builder, and an effective amount of proteolytic enzyme elaborated by T hermoactinomyces vulgaris ATCC 15734. The relative proportions of detergent builder and enzyme can be adjusted depending on the use for which the product is intended. Suitable compositions can, for example, be formulated with more or less builder to be especially adapted to light-duty or heavy-duty applications.

DETAILED DESCRIPTION OF THE INVENTION More specifically, in its broadest embodiment, the present invention relates to a detergent composition consisting essentially of a water-soluble synthetic detergent and an effective amount of a proteolytic enzyme elaborated by Thermoactinomyces vulgaris ATCC 15734. While the effective amount of said enzyme can be determined for any intended purpose and can therefore range over broad limits, it is preferred that the detergent compositions of this invention, including both built and unbuilt compositions, contain a level of protease activity in the range of 50 to 20,000 casein assay activity units per gram and preferably from to 15,000 casein assay activity units per gram. This level of enzymatic activity is sufficient to ensure that enzymatic activity will be at an effective level over a broad range of product compositions. Determination of casein activity, as that term is used in describing the present invention, is explained below. Compositions which contain less than 50 casein assay activity units per gram fail to provide an adequate aid to the overall cleaning process. There appears to be no advantage obtained in overall cleaning performance when 20,000 casein activity units per gram are exceeded.

A built detergent composition of the present invention consists essentially of a water-soluble synthetic detergent and a water-soluble builder selected from inorganic and and organic alkaline detergency builder salts, the proportion of detergent to builder ranging from 5:1 to 1:20, and preferably 2:1 to 1:10, and an effective amount of a proteolytic enzyme elaborated by Thermoactz'nomyces vulgaris ATCC 15734.

Examples of suitable detergent compounds which can be employed in accordance with the present invention include the following water-soluble anionic, nonionic, ampholytic, and zwitterionic synthetic detergents:

, (a) Water-solublesoaps-Examples of suitable soaps for use in this invention are the sodium, potassium, ammonium and alkanolammonium (e.g., mono-, di-, and triethanolammonium) salts of higher fatty acids (C -C Particularly useful are the sodium and potassium salts of the mixtures of fatty acids derived from coconut oil and tallow, i.e., sodium' and potassium tallow and coconut soaps.

(b) Anionic synthetic non-soap detergents.-A preferred class can be broadly described as the water-soluble salts, particularly the alkali metal salts, of organic, sulfuric acid reaction products having in their molecular structure an alkyl radical containing from about 8 to about 22 carbon atoms and a radical selected from the group consisting of sulfonic acid and sulfuric acid ester radicals. (Included in the term alkyl is the alkyl portion of higher acyl radicals.) Important examples of these anionic synthetic. detergents are the sodium or potassium alkyl sulfates, especially those obtained by sulfating the higher alcohols (C -C carbon atoms) produced by reducing the glycerides of tallow or coconut oil; sodium or potassium alkyl benzene sulfonates, in which the alkyl group can be a straightor branched chain and contains from about 9 to about 15 carbon atoms, preferably about 12 to 14 carbons; sodium alkyl glyceryl ether sulfonates, especially those esters of the higher alcohols derived from tallow and coconut oil; sodium coconut oil fatty acid monoglyceride sulfates and sulfonates; sodium or potassium salts of sulfuric acid esters of the reaction product of one mole of a higher fatty alcohol (e.g., tallow or coconut oil alcohols) and about 1 to 6 moles of ethylene oxide; sodium or potassium alkyl phenol ethylene oxide ether sulfates, with 1 to units of ethylene oxide per molecule and wherein the alkyl radicals contain from 8 to 12 carbon atoms; the reaction product of fatty acids esterified with isethionic acid and neutralized with sodium hydroxide where, for example, the fatty acids are derived from coconut oil; sodium or potassium salts of fatty acid amide of a methyl tauride in which the fatty acids, for example, are derived from coconut oil; sodium and ,potassium salts of 'SO- -sulfonated C -C a olefins.

(c) Nonionic synthetic detergents-One class of nonionic detergents can be broadly defined as compounds produced by the condensation of alkylene oxide groups (hydrophilic in nature) with an organic hydrophobic compound, which may bealiphatic or. alkyl aromatic in nature. The length of the hydrophilic or polyoxyalkylene radical which is condensed with any particular hydrophobic group can be readily adjusted to yield a water-soluble compound having the desired degree of balance between hydrophilic and hydrophobic elements. A second class of nonionic detergents comprises higher fatty amides. A third class of nonionic detergents has semi-polar characteristics. These three classes can be defined in further detail as follows:

(1) One class of nonionic synthetic detergents is marketed under the tradename of Pluronic. These detergent compounds are formed by condensing ethylene oxide with a hydrophobic base formed by the condensation of .propylene oxide with propylene glycol. The hydrophobic portion of the molecule which, of course, exhibits Water insolubility, has a molecular weight of from about 1500 to 1800. The addition of polyoxyethylene radicals tothis hydrophobic portion tends to increase the water=solubility of the molecule as a whole and the liquid character'of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product. 1

(2) Alkylphenol-polyethylene oxide condensates 'are condensation product of alkyl phenols having an alkyl group containing from about 6 to 12 carbon atoms-in either ,a straight-chain or branched-chain configuration with ethylene oxide, the said ethylene oxide being present in amounts equal to 5 to 25 moles of ethylene oxide p ale of alkyl t a The al y ubstitus t in such 4 compounds may be derived from polymerized propylene, diisobutylene, octene, or nonene, for example.

(3) Nonionic synthetic detergents can be derived from the condensation of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylene diamine and include compounds containing from about 40% to about 80% polyoxyethylene by weight and having a'molecular weight of from about 5,000 to about 11,000. Such compounds result from the reaction. of ethylene oxide .with a hydrophobic base constituted of the reaction product of ethylene diamine and excess 'propylene oxide, said "base having a molecular weight of the order of 2,500 to 3,000.

(4) Other nonionic detergents include condensation products of aliphatic alcohols having from 8 to 22 carbon atoms, in either straight chain or branched chain configuration, with ethylene oxide, e.g., a coconut alcoholethylene oxide condensate having from 5 to 30'mo1es of ethylene oxide per mole of coconut alcohol. 1

(5) The ammonia, monoethaenol and diethanolamides of fatty acids having an acyl moiety of from'about 8 to about 18 carbon atoms are useful nonionic detergents. These acyl moieties are normally derived from naturally occurring glycerides, e.g., coconut oil, palm oil, soybean oil and tallow, but can be derived synthetically, e.g.,.by the oxidation of petroleum, or by hydrogenation of carbon monoxide by the Fischer-Tropsch process.

) (6) Semi-polarnonionic detergents include long chain tertiary amine oxides corresponding to the following general formula wherein R is an alkyl radical of from about 8 to about 18 carbon atoms, R and R are each methyl, ethyl or hydroxyethyl radicals, R is ethylene, and n ranges from 0 to about 10. The arrow in the formula is a conventional representation of a semi-polar bond. Specific examplesof amine oxide detergents include dimethyldodecylamine oxide and bis(2-hydroxyethyl) dodecylamine oxide.

(7) Other semipolar nonionic detergents include long chain tertiary phosphine oxides corresponding to the fol lowing general formula RRRP O wherein R is an alkyl,falkenyl or monohydroxyalkyl radical containing from 10 fo 20 carbon atoms and R and R are eachfalkyl or monohydroxyalkyl groups containing from 1 to.,3 car bonatoins. The arrow in the formula is a conventional representation of a semi-polar bond. Examplesof suitable phosphine oxides are found in US. Pat. 3,304,263 which issued Feb. 14, 1967, and includes: dimethyldodecylphosph ige oxide and dimethyl-(Z-hydroxydodecyl) phosphine 0x1 e.

8) Still other semi-polar nonionic synthetic detergents include long chain sulfoxides having the formula wherein R is analkyl radical containing from about '10 to about 28 carbon atoms, from 0 to about 5 ether linkages and from 0 to about 2 hydroxyl substituents, at least one moiety of .R being an alkyl radical containing-0 ether linkages. andcontaining from about 10 to about 18 carbon atoms, and wherein R is an'alkyl radical containing from 1 to 3 carbon atoms and from onetto two hydroxyl groups. Specific examples of these sulfoxides'are: dodecyl methyl sulfoxide and. 3-hydroxy tridecyl methyl sulfoxide.

(d) Ampholytic synthetic detergents can be broadly described as derivatives of aliphatic secondary 'and tertiary amines,in which the aliphatic radical can .be straight chain or branched alkyls and wherein one of the aliphatic. substituents containsfrom about 8 to -l8'carbonatoms and one contains an anio ic w t r so ubilig g group, as,

carboxy, sulfo, sulfato, phosphate, or phosphono. Examples of compounds faling within this definition are sodium 3 dodecylaminopropionate and sodium-3-dodecylaminopropane sulfonate.

(e) Zwitterionic synthetic detergents can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium and sulfonium compounds, in which the aliphatic radical can be straight chain or branched alkyl, and wherein one of the aliphatic substituents contains from about 8 to 24 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfo, sulfato, phosphate, or phosphono. Examples of compounds falling within this definition are 3-(N,N-dimethyl- N-hexadecylammonio) propane-l-sulfonate and 3-(N,N- dimethyl-N-hexadecylammonio) 2 hydroxy propane-lsulfonate which are preferred for their cool water detergency characteristics. See, for example, Snoddy et al., Canadian Pat. 708,148.

Preferred organic detergents include sodium alkyl benzene sulfonate, sodium alkyl sulfate, and mixtures thereof wherein the alkyl group is of branched or straight chain configuration and contains about 10 to about 18 carbon atoms. Specific examples of preferred organic detergents include sodium decyl benzene sulfonate, sodium dodecyl benzene sulfonate, sodium tridecyl benzene sulfonate, sodium tetradecyl benzene sulfonate, sodium hexadecyl benzene sulfonate, sodium octadecyl sulfate and sodium tetradecyl sulfate.

These soap and non-soap anionic, nonionic, ampholytic and zwitterionic detergent compounds can be used singly or in combination. The above examples are merely illustrations of the numerous suitable detergents. Other organic detergent compounds can also be used.

DETERGENCY BUILDERS Suitable builders are water soluble inorganic alkaline builder salts, organic alkaline sequestering builder salts or mixtures thereof.

Suitable inorganic, alkaline builder salts include alkali metal carbonates, phosphates, polyphosphates and silicates. Specific examples of such salts are sodium and potassium tripolyphosphates, carbonates, phosphates and hexametaphosphates.

Suitable organic alkaline sequestering builder salts include alkali metal, ammonium and substituted ammonium polyphosphonates, polyacetates, and polycarboxylates.

The polyphosphonates specifically include the sodium and potassium salts of ethylene diphosphonic acid, sodium and potassium salts of ethane-l-hydroxy-1,1-diphosphonic acid and sodium and potassium salts of ethanel,1,2-triphosphonic acid. Other examples include the water-soluble [sodium, potassium, ammonium and substituted ammonium (substituted ammonium, as used herein, includes mono, di-, and triethanol ammonium cations)] salts of ethane-Z-carboxy 1,1-diphosphonic acid, hydroxymethanediphosphonic acid, carbonyldiphosphonic acid, ethane-l-hydroxy-l,1,2-triphosphonic acid, ethane-Z-hyd-roxy1,l,2-triphosphonic acid, propane-l,1,3,3-tetraphosphonic acid, propane-1,1,2,3-tetraphosphonic acid, and propane-1,2,2,3-tetraphosphonic acid.

Examples of the above polyphosphonic compounds are disclosed in US. Pats. 3,159,581, 3,213,030, 3,387,024, 3,400,148, 3,400,176, 3,400,151, 3,422,021, 3,422,137.

The polyacetate builder salts suitable for use herein include the sodium, potassium, lithium, ammonium, and substituted ammonium salts of the following acids: ethylenediaminetetraacetic acid, N-(Z-hydroxyethyl)-ethylenediaminetriacetic acid, N-(Z-hydroxyethyl) nitrilodiaeetic acid, diethylenetriaminepentaacetic acid, 1,2 diaminocyclohexanetetraacetic acid and nitrilotriacetic acid. The trisodinm salts of the above acids are generally and preferably utilized herein.

The polycarboxylate builder salts suitable for use herein consist of water-soluble salts of polymeric aliphatic polycarboxylic acids selected from the group consisting of (a) Water-soluble salts of homopolymers of aliphatic polycarboxylic acids having the following empirical formula:

[ 1 if (ilOOH I:

wherein X, Y, and Z are each selected from the group consisting of hydrogen, methyl, carboxyl, and carboxymethyl, at least one of X, Y, and Z being selected from the group consisting of carboxyl and carboxymethyl, provided that X and Y can be carboxymethyl only when Z is selected from carboxyl and carboxymethyl, wherein only one of S. Y, and Z can be methyl, and wherein n is a whole integer having a value within a range, the lower limit of which is three and the upper limit of which is determined by the solubility characteristics in an aqueous system;

(b) Water-soluble salts of copolymers of at least two of the monomeric species having the empirical formula described in (a), and

(c) Water-soluble salts of copolymers of a member selected from the group of alkylenes and monocarboxylic acids with the aliphatic polycarboxylic compounds described in (a), said copolymers having the general formula g (1 In) XL 6 0H m]n wherein R is selected from the group consisting of hydro gen, methyl, carboxyl, carboxymethyl, and carboxyethyl; wherein only one R can be methyl; wherein m is at least 45 mole percent of the copolymer; wherein X, Y, and Z are each selected from the group consisting of hydrogen, methyl, carboxyl, and carboxymethyl; at least one of X, Y, and Z being selected from the group of carboxyl and carboxymethyl providedthat X and Y can be carboxymethyl only when Z is selected from the group of carboxyl and carboxymethyl, wherein only one of X, Y, and Z can be methyl and wherein n is a whole integer within a range, the lower limit of which is three and the upper limit of which is determined primarily by the solubility characteristics in an aqueous system; said polyelectrolyte builder material having a minimum molecular weight of 350 calculated as the acid form and an equivalent weight of about 50 to about 80, calculated as the acid form (e.g., polymers of itaconic acid, aconitic acid; maleic acid; mesaconic acid; fumaric acid; methylene malonic acid; and citraconic acid and copolymers with themselves and other compatible monomers such as ethylene). These polycarboxylate builder salts are more specifically described in US. Pat. 3,308,067, issued Mar. 7, 1967 to Francis L. Diehl entitled Polyelectrolyte Builders and Detergent Compositions."

Mixtures of the above-described alkaline builder salts can be utilized to advantage in this invention.

Satisfactory results overall are obtained by built detergent compositions which contain .from 50 to 20,000 casein activity units per gram and preferably from to 15,000 casein activity units per gram.

Protease activity is described herein in terms of casein assay activity units. The casein assay method of determining protease activity units is well known. A detailed discussion appears in B. Hagihara et al., J. Biochem. (Tokyo), 45,185 (1958) and also in M. Kuntz, J. Gen. Physiol., 291 (1947). The casein assay procedure in general terms involves allowing a solution of an enzyme preparation to be evaluated to digest by hydrolysis a solution of a casein substrate at a given pH and temperature. At a certain point in time, the reaction is stepped by addition of trichloroacetic acid, the solution is filtered and the color of the filtrate containing the digested casein is developed employing Folin-Ciocalteau phenol reagent. The degree of enzymatic activity in terms of casein assay activity units per gram is determined by comparing the spec trophotometric response with that of solutions of varying concentrations of reagent grade tyrosine.

A pure proteolytic enzyme preparation elaborated by T hermoactinomyces vulgaris ATCC 15734 provides activity in the general range of 8,000,000 to 10,000,000 units per gram. This refers to the enzyme on a pure basis. Ordinarily, however, it is not necessary to take the additional costly steps to recover a pure enzyme. Typical and'standard recovery processes provide an enzyme preparation containing from 1 to 75% by weight of protease enzyme and the balance 25-99% comprising organic and inorganic materials including non-enzyme proteins, sodium sulfate, calcium sulfate, sodium chloride, calcium chloride, sodium hydroxide, unused carbonsource such as starch, water insolubles,'magnesium sulfate, potassium phosphate, ethyl alcohol, carbohydrates, lipids and color bodies. The active enzyme content of preferred enzyme preparations useful in this invention range from 3 to 50%, and the balance 50 to 97% being the inert salts which are introduced during the fermentation and recovery processes.

Ordinary'enzyme preparations elaborated by T hermaactinomyces vulgar-is ATCC 15734 have casein assay activity values ranging from 100,000 to 2,000,000 casein activity units per gram. It is evident, therefore, that in practicing the present invention, the amount or weight proportion of a given enzyme preparation in a detergent composition depends upon its potency in terms of casein assay activity units. The amount of enzyme which is added to a detergent composition is greater if an enzyme preparation is used which has an activity in the range of about 100,000 casein assay activity units per gram as compared, for instance, to an enzyme preparation which has an activity in the range of about 2,000,000 units per gram.

The primary objective, regardless of the specific activity units of an enzyme preparation, is to use an amount sufficient to accomplish the desired level of cleaning and stain removal. As noted previously, this level will generally be in the range of 5020,000 casein assay activity units per gram of detergent compositions.

The detergent compositions of the present invention can be formulated into solid or liquid compositions. Ap propriate solid forms include granular and powder compositions, flakes, tablets, bars and the like. As respects liquid formulations, they can be water and alcohol based. Hydrotropes, e.g., potassium toluene or xylene sulfonate, can be used to facilitate solubilities and ease of preparation.

Liquid compositions of the invention contain from 3% to 40%, preferably 5 to 30% water-soluble synthetic detergent or mixtures thereof, from 0 to 15% organic hydrotrope, preferably 2 to 12%, e.g., potassium toluene sulfonat-e, and 30 to 97% water, preferably 40 to 90% water, and an amount of proteolytic enzyme elaborated by T hermoactinomyces vulgaris ATCC 15734 to provide from 50 to 20,000, prefer-ably 100 to 15,000 caseinassay activity units per gram of detergent composition.

Unbuilt liquid detergent compositions such as lightdutydishwashing compositions ordinarily provide a washing solution having a pH near neutral, e.g.', 6.54.5. The compositions of this invention are highly useful under such conditions. They are especially useful, however, in an alkaline system ranging from 8 to 12, and optimum benefit is obtained in a range of 9.5 to 11.5. While most enzymes are denatured by such highly alkaline conditions, the proteolytic enzyme elaborated by Thermoactinomyces vulgaris ATCC 15734 is surprisingly stable and resistant to degradation. For this reason, a preferred embodiment of this invention is a highly alkaline built laundry composition containing a synthetic detergent, a builder, and the proteolytic enzyme elaborated by Thermoactir'tomyces vulgaris ATCC 15734 in the proportions described previously which composition provides in aqueous solution a pH in the range of 9.5 to 11.5. If the mixture of the esse tia g d e ts tai t p o ide a pH in'this range, additional alkaline agents can be added to the composition or directly to the washing solution.

In addition to the ingredients mentioned above, the present invention permits the optional presence of additives which are ordinarily added to complete detergent formulations. By way of examples, are perfumes, dyes, diluents, pH adjusters, anti-corrosion agents, anti-redeposition agents and the like. In addition, additional enzymes can be added to supplement or complement the proteolytic enzyme elaborated by Thermoactinomyces vulgaris ATCC 15734. Added enzymes can be proteases, amylases; lipases and the like. In addition, mixtures of such enzymes can be used in combination with theproteolytic enzyme elaborated by Thermoactinomyces vulgarz's ATCC 15734 according to this invention. Y

The composition of the present invention can be pre pared in any satisfactory method. An enzyme preparation, such as each of those prepared according to Preparation A and Preparation B below, can be mechanically mixed with a synthetic detergent or a spray-dried detergent comprising a detergent and a builder in the proportions noted above. To avoid segregation or separation of the fine enzyme powders, the composition of the present invention can be prepared according to the manner described in US. Pat. 3,451,935, granted June 24, 1969; According to that patent, an excellent process is described according to which an aqueous slurry of an enzyme preparation is sprayed onto an enzyme carrier granule which is comprised of a hydratable salt. The water from the enzyme slurry is adsorbed as Water of hydration and the enzyme is firmly retained on the surface of the enzyme carrier granule. These enzyme carrier granules can themselves constitute a detergent composition or such carrier granules can be added to a spray-dried granular detergent composition.

Having described the present invention in detail, it is now demonstrated by the following examples, preceded by two illustrative preparations of proteolytic enzyme elaborated by T hermoactinamyces vulgaris ATCC 15734.

PREPARATION A Spores on four slants of Soil Bennett starchagar were well kneaded with an inoculating loop. Five ml. of sterile suspending medium were added to each slant. The spores were removed frorrnthe agar surface with an inoculating loop. The spores from each two slants were combined in a sterile test tube. Spore suspensionswere effected by vortex agitation for 10 minutes. The two spore suspensions wereadded to a sterile bottle containing 10ml. of suspending medium and mixed by shaking.

SEED CULTURE PREPARATION I- ive ml. of spore suspension were inoculated into four, 4-lrterflasks-containing 0.5 liter of the following growth medium (themedium hadbeen previously sterilizedin an autoclave for. -30 minutes at 1 21 C.): v

Soluble potato starch 1 g. per liter..- 20.0 Defatted's'oybean hour 2 do 12.0

MgSO -7H O d0 0.50 K2HPO4 .dO v 4.0 KH P do 0.40 Na'lco 127 antifoam 3 ml. per liter "1.0

" sels byh ating. in a autoc aye to: 1 h rs at1121 The composition of the production medium is indicated below:

Soluble potato starch g. per 10 liters 200 Defatted soybean flour do 120 MgSO -7H do 5.0 Nalco-127 antifoam ml. per liters 10.0 Tap water .do 9800 A phosphate solution comprised of K HPO 40 g. and KH PO 4 g./200 ml. of distilled was autoclave separately for 20 minutes at 121 C., and added to each fermenter vessel immediately prior to inoculation. The final pH was 7.6.

After the temperature of the growth menstruurn reached 50 C., 500 ml. of seed inoculum were added to each fermenter vessel and the fermentation was carried out at 50 C. while air was introduced at the rate of 1 volume of air/volume of growth medium/ minute, while the medium was agitated at 300-350 r.p.m. The fermentation was terminated after 20 hours. The terminal pH was 6.8.

RECOVERY Growth was separated from the aqueous phase by centrifugation in a Sharples centrifuge at 10 C. The temperature of 16 liters of broth was brought to 4 C. and the pH adjusted to 7.2 with 1 N NaOH. Decolorization was efiected by the addition of Cationic F -50 (alkyldimethylbenzene ammonium chloride, Nippon Yushi Co., Japan) to a final concentration of 0.3% and centrifugation in a Sharples centrifuge. Hy-flo super cel was added to the supernatant which was then thrice filtered through a Buchner funnel, employing Reeve Angel No. 230 filter paper coated with a thin layer of celite analytical filter aid (Johns-Manville). Denatured ethanol was cooled to 10% in a Dry Ice acetone bath and added to precooled (4 C.) filtrate to a final concentration of 75%. The solvent-supernatant mixture was agitated for minutes. The precipitate was separated from the aqueous phase by passage through a Sharples centrifuge. The solids were quick frozen in Dry Ice and lyophilized. The dried enzyme (88 g.) was homogenized in a mortar and pestle and filtered through a Tyler Sieve with a porosity of 80 microns. Sixty grams of the dried enzyme were dissolved in 200ml. of distilled water. The pH was adjusted to 5.5 which concentrated HCl. The enzyme solution was cooled to 4 C. and fractionally precipitated with precooled (--10 C.) denatured ethanol at 30%, 58%, and 75%. The solids were separated from the aqueous phase by centrifugation at room.- temperature, and lyophilized. By this fractional precipitation procedure, a total 77.7% of the original activity was recovered; the 75% alcohol fraction contained 60.2% of the original activity. The enzyme recovered from the 30% alcohol precipitation step had 57,400 casein assay activity units per gram; the enzyme preparation recovered from the 60% alcohol precipitation step had 58,800 casein assay activity units per gram; and the 75 alcohol precipitation step had 970,000 casein assay activity units per gram.

PREPARATION B Fermentation Spores on two slants of Soil Bennett Starch Agar were well kneaded with an inoculating loop. Nine ml. of suspending medium were added to each slant. The spores were removed from the agar surface with an inoculating loop, and dispensed into two sterile test tubes. Spore suspensions effected by vortex agitation for 10 minutes. The two suspensions were added to a sterile test tube.

Seed culture preparation One ml. of spore suspension was inoculated into twenty, 500 ml. Erlenmeyer flasks containing 40 ml. of the following growth medium (the medium had been previously sterilized in an autoclave for 20 minutes at 121 C.):

Tap water to 40 ml.

.1. T. Baker Chemical Company, Phllllpsburg, NJ.

Ntsshln Selyu Co., Ltd., Tokyo, Japan.

The flasks were incubated on a reciprocating shaker at r.p.m. and 50 C. for 16-20 hours.

Recovery The mycelium was sedimented by centrifugation at 7,000 r.p.m. at room temperature for 15 minutes. The supernatants from three successive fermentations were combined and cooled to 4 C. The pH was adjusted to 6.8 with 1 N NaOH. Decolorization was effected by the addition of cationic Fg-SO (alkyldimethylbenzene ammonium chloride, Nippon Yushi Co., Japan), to a final concentration of 0.3% Hy-flo Super Cel was added to the decolorizing mixture to a concentration of 2-3%. The mixture was slowly agitated for 3 minutes and then permitted to stand undisturbed for 10 minutes. The mixture was twice filtered through a Buchner funnel, employing Reeve Angel No. 230 filter paper coated with a thin layer of celite analytical filter aid (Johns-Manville). The filtrate was then filtered through a vacuum funnel using Whatman No. 40 filter paper. The pH of the filtrate was adjusted to 5.5 with l N HCl. Denatured ethanol was cooled to -10 C. in Dry Ice acetone bath and added to precooled (4 C.) filtrate to a final concentration of 75%. The mixture was stirred for 5 minutes and then placed at 4 C. overnight. Most of the supernatant was decanted, the remainder was filtered with No. 230 filter paper. The solids which collected on the filter paper were removed and lyophilized. The proteolytic enzyme had a casein assay activity value of 471,000 activity units per gram.

The proteolytic enzyme elaborated by Thermzoactinomyces vulgaris ATCC 15734, according to Preparation B, was evaluated in a detergent composition embodying preferred embodiments of the present invention. This procedure is described below under Stain Removing Tests are part of Example I, together with results demonstrating the unique stability of the proteolytic enzyme especially under highly alkaline hot water washing conditions in a detergency system.

EXAMPLE I Stain removing tests Muslin swatches were stained by passing strips of muslin through a padding bath containing the staining solution, passing the muslin through wringers and a drying oven. The stained strip was cut into 5" x 5%" swatches. These swatches were laundered in an automatic miniature washer at 125 F. in water of 7 grain hardness for 10 minutes. Each composition being tested was used to wash a soiled load consisting of 3 swatches each of (1) gravy (a substrate sensitive to both proteolytic and amylolytic activity), (2) spinach (a substrate primarily sensitive to proteolytic activity), and (3) milk substitute (a substrate primarily sensitive to proteolytic activity), and (4) licorice (a substrate primarily sensitive to amylolytic activity) stained muslin in the presence of two untreated terrycloth swatches added to provide bulk to the washload.

The test or control composition used for a comparison was a conventional built anionic-containing detergent formulation and was employed in an amount of 6.75 grams/1V2 gal. water (equivalent to 1 cup/ 17 gal. water). The enzyme to be evaluated was added in the form of a water solution to provide the desired level of enzyme. The swatches were washed, dried, and ironed and their white- 11 ness levels were measured employing a Hunter Color- Ditference Meter. The model employed was Model D25 from Hunter Associates Laboratory, Fairfax, .Va. This device operates on the principle of reflectance and meas- The composition is expecially useful in an aqueous solution having a pH 7.5.

' Percent Ethoxylated tallow fatty alcohol (condensation product ures degree of whiteness. The greater the Hunter value, of 3 moles ethylene oxide and one mole tallow alcothe greater the whiteness level. The stain-removing effect hol) 95 of an enzyme is determined by comparing the Hunter Proteolytic enzyme elaborated :by Thermoactinomyces Whiteness values obtained by compositions with and withvulgaris ATCC 15734 having 20% active enzyme 5 1. casein assay activity units per gram.

Ingredient: Parts by weight EXAMPLE 111 A mixture of 55% sodium tallow alkyl sulfate A and 45% Sodium linear alkyl benzene Sulfo granular pre-soakmg composition etfectrve 1n the renate wherein the alkyl chain distribution is inoval of pilneuiaceous and Siarch stains has the follow 16% C11 27% C12 35% C13, and 22% C14 175 mg composition in parts by weight. Percent ;322:3 g'g gagz g'gi Ethoxylated tallow fatty alcohol (3 moles ethylene 1.8:1 6:0 oxide/mole of alcohol) 4.0 Coconut fatty acid ammonia amide 2.5 S i tnpolyphosphate Sodium sulfate '14.0 odlum Rerborate Water Proteolytlc enzyme elaborated by T hermoactinomyces vulgaris ATIC 15734 having 50% active Results of these stain tests are tabulated in Table I. en 2,0 The stability of an enzyme elaborated from Thermoactino- Water 9 0 myces vulgaris ATCC No. 15734 in a highly alkaline detergency system is clearly demonstrated. This is a valu- In Q a1d.the pubhc m the pracnce the presiant able discovery for it affords an enzyme for detergent manmventlon pomted out i a culture mlcroorgamsm ufacture which can be usefully incorporated into highly Thqrmoactmamwces vulga.ns IS on deposlt at and frely alkaline built detergent compositions which provide opavallaible from the American Culture i l timum cleaning results in Washing Solutions having a PH Washington, DC. It has been assigned the designation in the range of 8-12 and preferably 9.511.5. Many ATCC enzymes, which have activity in laboratory tests at lower All percentfges proportions herem are by weight pHs, are denatured at the relatively severe pH conditions unless .otherwlse. specified encountered in built detergent compositions and solutions Havmg (described. the prfasem mvegnon m detall containing such compositions illustrated ts effectiveness 111 a practical demonstration, As respects data tabulated for the gravy stain, a difierwhat 15 clalmed ence of about 2 Hunter |Whiteness units is a visually ob- A detergent compfsltm conslstmg essentlaliy Servable difierence; with spinach Stains, an observable of a water-soluble, synthetic detergent and of proteolytic difference is about 4 units; with milk stains, about 1.8 enzyme elaborated y T a tinvmyces vulgaris ATCC units; and with licorice stains, about 2 units. The de- 40 15734 said COmPQSiFiOH having a PH of from to naturing effect of the higher pH (10.2) on th i i nd a protease activity of from 50 to 20,000 casein units of the subtilisin is evident by comparing the stain-remov- Per ing values at PH with PH In each instance the 2. A built detergent composition consisting essentially enzyme elaborated from Thermoaclinomyces vulgaris of a Water-soluble, synthetic detergent and a water-soluble ATCC 15734 sustained its valuable activity thereby dembullder seflected from morgamc F Orgamc alkalme deter onstrating its capability to resist denaturation' by hot bullder Salts the Propomon of detergent to builder water and highly alkaline systems, as well as such typical a i from 511 to 1120, and a proteolytic enzyme additives to built detergent compositions as anionic syn= eliiborated by hermoflcelinomyces vulgal'is ATCC 15734, thetic detergents, builders, silicates, sulfates, amides and Sand compositlfm havlng 3 P of from t0 and a water. While useful enzymatic activity is provided by the Protease actlvlty Of'from 50 20,000 casein units per enzyme of the present invention at lower pHs, e.g., 9.4, grama preferred way of practicing the invention is with de- A built detergent composition of Claim ipnwhich tergency systems employing higher H i 95 t 115, the proportion of detergent to builder is in the range of although practical benefits are obtained throughout the 1 t0 I pH rangeof about 8.0 to 12.0. 4. A built detergent composition of claim 3 in which Gravy Spinach 'Milk Licorice Weight stain stain stain stain percent enzyniire Hunter whiteness, pH of Enzyme derived fromproduct 9.4 10.2 9.4 10.2 9.4 10.2 9.4 10.2

Bacillus subtilus derived Carlsberg strain of subtilisin 0.0115 26.0 12.0 25.7 6.3 16.0 10.2 2.5 4.3 Thermoactinomyces vulgaris (Protease TV-ATCC No. 1573 0.0115 2 1.0 25.0 18.0; 20.4 10.2 14.4 1.0 6.0

1 Product usage was 6.75 g./l.' gal. water (equivalent to 1 cup/17 ga1. water), providing a concentration of enzyme in solution of about 3 p.p.m.

, The Carlsberg strain is a known subtllisin strain, the amino acid sequence of which is described in Smith et al., The CompleterAmino Acid Sequence of Two Types of Subtilisin, BPN and Carlsberg," J ..oi .Boil. Chem., vol. 241, Dec. 25, 1966 at p. 5974. This subtilisin strain is characterized by a tyrosine to tryptophan ratio of about 13 t0 1. The above reference including its description of the amino acid sequence of the Carlsberg subtilisin is hereby incorporated by reference. 7

EXAMPLE II the detergent is an anionic synthetic detergent and the builder is selected from the group consisting of sodium tripolyphosphate, sodium carbonate, trisodium ethylenediarninetetraacetic acid, and trisodium nitrilotriacetic acid.

(References on following page) 14 References Cited Amylolytic Strains of Thermoaclinomyces Vulgaris, by UNITED STATES PATENTS K110 et 611., 'vol. 92, pp. 723-726, J. Bacteriology (1966);

Alkali-Resistant Enzyme for Detergents, by Green, Soap 3,451,935 6/1969 Roald et a1 252-135 3,574,120 4/1971 Siebert et a1. fin 252 135 X 5 igglhem. Spec1alt1es, May 1968, pp. 86, 88, 90, 94, and 3, 97 M y 9 fiesai et a1 P ifi ti and Pro pertles of Proteolyuc 3655570 4/1972 Isono et a1 252*132 Enzymes from Thermophilic Actinomycetes, Journal of OTHER REFERENCES Bacteriology, October 1969, pp. 149-155.

The Actinomycetes, vol. 2 by S. A. Waksman, p. 309, Williams-Wilkins C0. (1961); Studies on the Proteolytic LEON ROSDOL Exammer Enzymes of Thermophilic Streptomyces by Mizusawa at SILVERSTEIN, Assistant E a al., pp. 884-895, vol. 28, No. 12, Agr. Biol. Chem.

(1964); Applied Microbiol., Vol. 9 1961), Studies 011 the Thermophilic Actinomycetes, pp. 394-399; Chem. 546 Abstr., V01. 70 (1969), p. 100, 112590; Isolation of 15 

